The goal of this research effort is to understand how various types of white blood cells recognize and respond to the presence of a microorganism or cancer cell in the body, or inappropriately recognize a normal component of the body (an auto-antigen). Our experiments are designed to provide a detailed understanding of how the substances (antigens) making up microorganisms, cancer cells, or normal self-components, are made visible to the defending cells of the innate (anti-unspecific) or adaptive (antigen-specific) limbs of the host defense system cells and how recognition of infections, malignant cells, or adjuvants is linked through complex cell-cell interactions to the induction of protective or self-destruction effector responses. We are now conducting studies at the cell and tissue level to relate the physiology of antigen and pathogen recognition to the development of effector function, immune memory, or tolerance. During the past year we have used these novel imaging methods in concert with classical cellular immune assays to extend our previously published work on the molecular mechanism by which the small adapter protein SAP controls sustained interaction of antigen-specific T and B-cells. Our studies revealed that SAP is key to lymphoid-lymphoid cell and not lymphoid-myeloid cell interactions, based on SLAM-family adhesive interactions involving CD84 and Ly108. Our findings showed that in contrast to T-dendritic cell interactions, T-B cell interactions were less integrin and more SLAM dependent after 10-15 minutes of interaction, explaining why we found that SAP-deficient T cells fail to adhere to B cells long enough to deliver help function. These findings have provided a new level of insight into molecular control of T cell-dependent humoral immune responses of the type that are critical for effective vaccine responses. In a second major effort, we extended our study combining intravital imaging, in vitro migration analysis, and flow cytometry, along with micro-CT and related tools for assessing bone structure, to explore the role of the lipid-signaling pathway involving S1P and its receptors S1P1 and S1P2 in control of osteoclastogenesis. Osteoclasts are large multinucleate myeloid cells responsible for bone resorbtion. They form by fusion of bone surface-adherent osteoclast precursors that are cells in the monocytoid series. We previously showed that S1P and S1P1 played a role in regulating the efficiency with which osteoclast precursor myeloid cells remained attached to the bone surface for long enough to participate in osteoclast formation, with S1P1 signaling promoting the return of the precursors to the blood before maturation occurs. We now have evidence that S1P2 acts in the opposite fashion, promoting chemorepulsion and augmenting the egress of the precursors from the blood and movement towards the bone surface. However, S1P2 operates in a very different range of S1P concentrations from S1P1, allowing these two receptors and the gradient of S1P that exists between bone surface and blood to finely regulate the rate of movement of osteoclast precursors to and from the bone surface, where other signals involving chemokines and integrins contribute to the rate of osteoclast formation. This study thus provides novel insight into a pathway with a major role in the balance of bone formation and destruction and points to a new target for interference with pro-osteoporotic processes. We are using our 2 photon intravital imaging technology on a range of other projects investigating the intersection of the immune system with vaccines, infectious antigen sources, and dying cells in tissues. We are extending our earlier studies on the chemokine control of CD4-CD8 T cell interactions to better understand how these cell types collaborate in lymphoid tissue to generate optimal primary and memory cell-mediated immune responses. Our recent work suggests that different dendritic cells initially present antigen to the majority of CD4 vs. CD8 T cells, raising questions about where and when during the response the two cell types interact with the same antigen presenting cell. We have also acquired data on the role of Tregs in controlling effector vs. central memory cell formation, the site(s) of delivery of TLR-conjugate vaccines to diverse dendritic cell populations in draining lymph nodes, and on the differential location of nave vs. memory cells in lymphoid tissues. The latter studies have also led to exciting new data on the highly localized positioning of a series of innate and adaptive effector populations within lymph nodes near to the subcapsular sinus. Macrophages lining this sinus play a critical first line defensive role against the spread of lymph-borne pathogens and these effectors appear pre-positioned to back up this initial defense against systemic spread of infections. We have also invested in expanding our imaging technologies by developing methods that permit performing 7 or 8 color fluorescent immunohistochemistry on lymphoid and other tissues, collecting tiled images across entire tissue sections, and computationally analyzing the data to assign multiple stains to specific cells in a multidimensional solid-phase analog of flow cytometric phenotyping. This method is showing us the complex and heterogeneous, non-random distribution of dendritic cell subsets in lymphoid tissues and the differences in how these cells capture antigen and present it to different T cell subsets. We are also developing new computer algorithms that permit not only the analysis of cell positioning, but a quantitative analysis of phenotypically complex cell populations within intact tissue (a procedure we call histo-flow for its similarity in outcome to flow cytometric analysis of dissociated cell populations). This methodology has potential applicability to human and NHP tissues and we have initiated collaborations to study follicular helper cell (Tfh) in infected to immunized monkeys, work that should contribute to the improvement of vaccines. We are also examining the role of various innate receptors in signaling to the cells we are imaging, attempting to integrate the actions of microbial stimuli in our assessment of immune cell behavior in situ. This work includes a broad analysis of NLR-family receptor function, along with use of traditional TLR ligands as adjuvants. With respect to innate cells, we have developed a novel method of cell transfer that permits the rapid screening of control vs. mutant (knock-out) cells for differences in in vivo behavior within a tissue site, especially the localized swarming response to focal tissue damage induced by laser heating. Preliminary studies using this advanced method have revealed a very specific role for lipid mediators in controlling neutrophil responses to cell death as well as an unsuspected effect of neutrophil aggregation on local tissue matrix structure. These studies are being extended further using 3D culture systems and microfabricated devices that permit precise control of chemoattractant gradient steepness and magnitude, as well as change in effective concentration over time. Using these 3D systems, we have developed an entirely new picture of the factors controlling persistent migration of dendrite cells that stands in marked contrast to the models derived from 2D migration studies. These latter experiments are also being complemented with collaborative work using a novel form of high resolution 3D isotropic imaging developed by the Betzig lab at Janelia Farms, HHMI, and high speed, high resolution studies using labeled dendritic cells and collagen fibers, to examine the mechanical forces that permit integrin-independent migration of these cells.